Social stress is as effective as physical stress in reinstating morphine-induced place preference in mice B. Ribeiro Do Couto, M. A. Aguilar, C. Manzanedo, M. Rodriguez-Arias, A. Armario, J. Minarro B. Ribeiro Do Couto, M. A. Aguilar, C. Manzanedo, M. Rodriguez-Arias, A. Armario, J. Minarro

Introduction Human heroin addicts that have been clean for years relapse when 1.Confronted with context or cues associated with heroin (old hangout, paraphernalia, etc.) 2.Given heroin or opportunity to use heroin 3.Experience stressful life events Human heroin addicts that have been clean for years relapse when 1.Confronted with context or cues associated with heroin (old hangout, paraphernalia, etc.) 2.Given heroin or opportunity to use heroin 3.Experience stressful life events

In animal studies when trained to self-administer or conditioned for a place preference and then undergo extinction will relapse also when 1. Presentation of drug associated cues (light, odor, drug paired chamber, etc.) 2.Exposure to the drug itself (injection) 3.Exposure to a stressor (foot shock, restraint, SD,etc.) In animal studies when trained to self-administer or conditioned for a place preference and then undergo extinction will relapse also when 1. Presentation of drug associated cues (light, odor, drug paired chamber, etc.) 2.Exposure to the drug itself (injection) 3.Exposure to a stressor (foot shock, restraint, SD,etc.) Introduction

Timeline of extinction-reinstatement model of self-administration Timeline of extinction-reinstatement model of self-administration Introduction Chambers & Self (2002) Neuropsychopharmacol. 27:889-5

 “Using the self-administration paradigm it has been reported that stress can be even more effective in reinstating drug seeking behavior than re-exposure to drugs.”  Shaham, etal Psychopharmacol. 119:  “Using the self-administration paradigm it has been reported that stress can be even more effective in reinstating drug seeking behavior than re-exposure to drugs.”  Shaham, etal Psychopharmacol. 119: Introduction

Self-administration of opioids and extinction-reinstatement experiments reveal that reinstatement is demonstrated by 1.Food deprivation 2.Administration of CRF 3.Not restraint stress Self-administration of opioids and extinction-reinstatement experiments reveal that reinstatement is demonstrated by 1.Food deprivation 2.Administration of CRF 3.Not restraint stress Introduction

Using conditioned place preference (CPP) procedure of extinction-reinstatement, Foot shock: 1.Delays the extinction of morphine CPP 2.Reinstates morphine CPP after extinction 3.Reactivates morphine seeking following drug free periods even when not exposed to extinction conditions Using conditioned place preference (CPP) procedure of extinction-reinstatement, Foot shock: 1.Delays the extinction of morphine CPP 2.Reinstates morphine CPP after extinction 3.Reactivates morphine seeking following drug free periods even when not exposed to extinction conditions Introduction

Psychological distress and provoking relapse to illicit drug use In humans, not only subordination stress is responsible for relapse Emotional stressors are the primary activators of social stress Psychological distress and provoking relapse to illicit drug use In humans, not only subordination stress is responsible for relapse Emotional stressors are the primary activators of social stress Introduction

“Social defeat in rodents could be considered as a stressor with essential ethological relevance” Previous studies reveal that morphine-induced CPP can be influenced by prior social defeat “Effects of social defeat on reinstatement of morphine seeking have not been studied” “Social defeat in rodents could be considered as a stressor with essential ethological relevance” Previous studies reveal that morphine-induced CPP can be influenced by prior social defeat “Effects of social defeat on reinstatement of morphine seeking have not been studied” Introduction

1.Evaluate the effects of two physical stressor (restraint and tail pinch) on the reinstatement of morphine-induced CPP 2.Determine the effects of a social defeat on reinstatement of morphine-induced CPP 3.Compare the effects of physical and social stressors on reinstatement of morphine- induced CPP 1.Evaluate the effects of two physical stressor (restraint and tail pinch) on the reinstatement of morphine-induced CPP 2.Determine the effects of a social defeat on reinstatement of morphine-induced CPP 3.Compare the effects of physical and social stressors on reinstatement of morphine- induced CPP Objectives of this Study

 OF1 strain of mice, 42 days old  Housed in groups of 4 for 10 days before training  All animals lived in a vivarium (21ºC)  Reversed light schedule (animals were tested when they are normally active)  Procedures in accordance with the European Communities Council Directives  OF1 strain of mice, 42 days old  Housed in groups of 4 for 10 days before training  All animals lived in a vivarium (21ºC)  Reversed light schedule (animals were tested when they are normally active)  Procedures in accordance with the European Communities Council Directives Methods

Conditioned Place Preference Examples

 8 identical Plexiglas boxes were used  Black and side were separated by a gray central compartment  Black side had fine grid floor (preferred?)  White side had wide grid floor  4 beams in each black and white and 6 in central area recorded position of mouse  8 identical Plexiglas boxes were used  Black and side were separated by a gray central compartment  Black side had fine grid floor (preferred?)  White side had wide grid floor  4 beams in each black and white and 6 in central area recorded position of mouse Methods

Pre-Conditioning (Pre-C) Manzanedo et al., 2001  Mice given access to both compartments for 900 s (15 min) for 3 days  Recorded on 3 rd day to determine unconditioned place preference  Animals that showed aversion (<33%) or preference (<66%) for any compartment were discarded (n=10) Pre-Conditioning (Pre-C) Manzanedo et al., 2001  Mice given access to both compartments for 900 s (15 min) for 3 days  Recorded on 3 rd day to determine unconditioned place preference  Animals that showed aversion (<33%) or preference (<66%) for any compartment were discarded (n=10) Methods

Pre-C notes  In each group half of animals were assigned morphine or control injection in black compartment, while the other half were assigned the injection in white compartment  No significant differences between in time spent in the drug-paired and vehicle paired compartments during Pre-C Pre-C notes  In each group half of animals were assigned morphine or control injection in black compartment, while the other half were assigned the injection in white compartment  No significant differences between in time spent in the drug-paired and vehicle paired compartments during Pre-C Methods

Second phase  4 days of conditioning to each group’s dose  Mice first received saline injection in the saline paired compartment for an amount of time dependent on dose  4 hour interval (home cage?)  Mice received morphine injection before confinement to drug-paired compartment  Central area was closed Second phase  4 days of conditioning to each group’s dose  Mice first received saline injection in the saline paired compartment for an amount of time dependent on dose  4 hour interval (home cage?)  Mice received morphine injection before confinement to drug-paired compartment  Central area was closed Methods

Third phase = Post-C  Test the rat for preference by removing guillotine doors on 8th day  Place rat in CPP enclosure for 15 min  Recorded which chamber rat preferred.  Only animals conditioned with morphine presented CPP in all experiments. Third phase = Post-C  Test the rat for preference by removing guillotine doors on 8th day  Place rat in CPP enclosure for 15 min  Recorded which chamber rat preferred.  Only animals conditioned with morphine presented CPP in all experiments. Methods

Extinction  Daily extinction sessions (15 min) were performed  When the time spent in the drug-paired compartment was not significantly different from Pre-C levels = extinction.  Apparently all animals received the same number of extinction session independent of individual scores.  Saline conditioned groups only went through one extinction session to confirm lack of CPP.  Morphine-conditioned mice with weak CPP (>60 s) were discarded. Extinction  Daily extinction sessions (15 min) were performed  When the time spent in the drug-paired compartment was not significantly different from Pre-C levels = extinction.  Apparently all animals received the same number of extinction session independent of individual scores.  Saline conditioned groups only went through one extinction session to confirm lack of CPP.  Morphine-conditioned mice with weak CPP (>60 s) were discarded. Methods

Reinstatement  Reinstatement sessions were the day after the last extinction session  Respective stress protocol was administered in the vivarium  Mice were put back into the CPP chamber with doors open (15 min)  This occurred immediately after stress or was delayed for 15 min. Reinstatement  Reinstatement sessions were the day after the last extinction session  Respective stress protocol was administered in the vivarium  Mice were put back into the CPP chamber with doors open (15 min)  This occurred immediately after stress or was delayed for 15 min. Methods

Table 1

Effects of restraint on the reinstatement of morphine-induced CPP  Extinction sessions (3-10 sessions) and after 24 h after extinction  15 min of immobilization-induced stress  impossible for mouse to turn around  Immediately or 15 min after (delayed) restraint stress, the reinstatement test was performed. Effects of restraint on the reinstatement of morphine-induced CPP  Extinction sessions (3-10 sessions) and after 24 h after extinction  15 min of immobilization-induced stress  impossible for mouse to turn around  Immediately or 15 min after (delayed) restraint stress, the reinstatement test was performed. Experiment 1

Figure 1 - Immediate

Figure 1 – Delayed Reinstatement

Table 1

Effects of tail pinch on morphine-induced CPP  One day after the last extinction session, mice were submitted to a modified tail-pinch schedule (15 min) to evaluate physical/tactile stress.  Tail-pinch performed with a plastic clothespin (800 g pressure)  Fastened on tail at cm from body  Performed in different cage  Immediately or 15 min after (delayed) tail-pinch, the reinstatement test was performed. Effects of tail pinch on morphine-induced CPP  One day after the last extinction session, mice were submitted to a modified tail-pinch schedule (15 min) to evaluate physical/tactile stress.  Tail-pinch performed with a plastic clothespin (800 g pressure)  Fastened on tail at cm from body  Performed in different cage  Immediately or 15 min after (delayed) tail-pinch, the reinstatement test was performed. Experiment 2

Figure 2 – Immediate

Figure 2 – Delayed Reinstatement

Effects of an agonistic encounter on the reinstatement of morphine-induced CPP  24 h after the last extinction session, mice were socially stressed by an aggressive opponent of equal age and body weight for 15 min  Aggressive opponent was selected for high aggression  Experimental mice were defeated in < 30 s, because opponent initiated attack immediately after seeing experimental mouse.  Defeat posture was an upright submissive position with limp forepaws, upwardly angled head, and retracted ears. Effects of an agonistic encounter on the reinstatement of morphine-induced CPP  24 h after the last extinction session, mice were socially stressed by an aggressive opponent of equal age and body weight for 15 min  Aggressive opponent was selected for high aggression  Experimental mice were defeated in < 30 s, because opponent initiated attack immediately after seeing experimental mouse.  Defeat posture was an upright submissive position with limp forepaws, upwardly angled head, and retracted ears. Experiment 3

Agonistic encounter without social defeat  Opponents were made temporarily anosmic by intranasal lavage with 4% zinc sulfate solution 24 h before encounter (Smoothy et al., 1986)  These mice elicit attack but never initiate attack  No aggressive behaviors were observed  These controlled for the extraneous variable of a social interaction.  Furthermore, another control was exposure to the cage without any social interaction. Agonistic encounter without social defeat  Opponents were made temporarily anosmic by intranasal lavage with 4% zinc sulfate solution 24 h before encounter (Smoothy et al., 1986)  These mice elicit attack but never initiate attack  No aggressive behaviors were observed  These controlled for the extraneous variable of a social interaction.  Furthermore, another control was exposure to the cage without any social interaction. Experiment 3

Table 1

Figure 3 – Social Defeat immediate reinstatement

Figure 3 – Social Defeat Delayed Reinstatement

Figure 4 –Non aggressive Social interaction

Figure 4 – Social interaction Delayed Reinstatement

 Immediate and delayed (30 min after) obtention of blood sample.  Immediate CORT release after stress is not a reflection of the actual stressor or of ACTH release.  This is because adrenocortical synthesis is saturated.  Severe stressors are characterized by a slower return to basal hormone levels.  Corticosterone levels were determined using a radioimmunoassay.  Immediate and delayed (30 min after) obtention of blood sample.  Immediate CORT release after stress is not a reflection of the actual stressor or of ACTH release.  This is because adrenocortical synthesis is saturated.  Severe stressors are characterized by a slower return to basal hormone levels.  Corticosterone levels were determined using a radioimmunoassay. Assessment of corticosterone Concentration

Corticosterone levels after stress exposure Stressed groups presented higher CORT levels than controls. Animals exposed to social defeat had higher levels of CORT than animals exposed to restraint or tail pinch in the 30 min groups. Defeat stress causes CORT increase to remain elevated?

 Physical stressors (restraint, tail pinch) administered immediately or 15 min before reinstatement tests are capable of reinstating CPP after extinction.  In general agreement with foot shock results.  Psychological stress (social defeat) suffered immediately or 15 min before reinstatement tests are capable of reinstating CPP after extinction.  Social stress is as effective as physical stress in reinstating drug-seeking behavior.  Physical stressors (restraint, tail pinch) administered immediately or 15 min before reinstatement tests are capable of reinstating CPP after extinction.  In general agreement with foot shock results.  Psychological stress (social defeat) suffered immediately or 15 min before reinstatement tests are capable of reinstating CPP after extinction.  Social stress is as effective as physical stress in reinstating drug-seeking behavior. Summary

 Pavlov first described the reinstatement of learned behaviors after extinction in his classical conditioning studies with dogs.  Social defeat induces activation of the mesocorticolimbic system which is involved in social stress-induced reinstatement.  Social defeat stress also increases µ-opioid receptor mRNA in the VTA, also involved in stress-induced reinstatement.  Pavlov first described the reinstatement of learned behaviors after extinction in his classical conditioning studies with dogs.  Social defeat induces activation of the mesocorticolimbic system which is involved in social stress-induced reinstatement.  Social defeat stress also increases µ-opioid receptor mRNA in the VTA, also involved in stress-induced reinstatement. Discussion

 Both physical stressors and social defeat increase extracellular levels of DA in the nucleus accumbens and prefrontal cortex.  Foot shock induces less DA release than drug priming but can be a more effective stimulus for reinstatement.  Stressors may induce a withdrawal-like state that lead to relapse.  But, withdrawal from heroin does not reinstate heroin seeking as stressors do.  Both physical stressors and social defeat increase extracellular levels of DA in the nucleus accumbens and prefrontal cortex.  Foot shock induces less DA release than drug priming but can be a more effective stimulus for reinstatement.  Stressors may induce a withdrawal-like state that lead to relapse.  But, withdrawal from heroin does not reinstate heroin seeking as stressors do. Discussion

 Stress may provoke relapse by interfering with neuronal inhibitory processes, which inhibits responding when reinforcers are not available.  Foot shock stress increases resistance to extinction.  Footshock’s reinstating effects depend on the medial septum, brain area involved in response inhibition.  Stress may provoke relapse by interfering with neuronal inhibitory processes, which inhibits responding when reinforcers are not available.  Foot shock stress increases resistance to extinction.  Footshock’s reinstating effects depend on the medial septum, brain area involved in response inhibition. Discussion

 Repeated exposure to drug might sensitize or induce neuroadaptations in brain systems involved in the stress response.  Foot shock stress increased CRF levels in the VTA and blockade of CRF receptors in this area attenuated the stress-induced reinstatement of cocaine seeking.  Experiments in this area are limited.  Neuroadaptation factors (BDNF, MAPK signal transduction, glutamate receptors) have yet to be explored in stress- induced reinstatement experiments.  Repeated exposure to drug might sensitize or induce neuroadaptations in brain systems involved in the stress response.  Foot shock stress increased CRF levels in the VTA and blockade of CRF receptors in this area attenuated the stress-induced reinstatement of cocaine seeking.  Experiments in this area are limited.  Neuroadaptation factors (BDNF, MAPK signal transduction, glutamate receptors) have yet to be explored in stress- induced reinstatement experiments. Discussion

 One study (foot shock, restraint, heroin) suggested that the effects of stress on reinstatement are context and time dependent.  This study shows contradictory results.  Restraint and tail pinch were administered in a different environment from CPP conditioning.  2 temporal intervals (0,15 min) before reinstatement test.  Here, all mice produced a clear reinstatement in morphine- induced CPP.  One study (foot shock, restraint, heroin) suggested that the effects of stress on reinstatement are context and time dependent.  This study shows contradictory results.  Restraint and tail pinch were administered in a different environment from CPP conditioning.  2 temporal intervals (0,15 min) before reinstatement test.  Here, all mice produced a clear reinstatement in morphine- induced CPP. Discussion

 An encounter with a non-aggressive opponent does not reinstate CPP.  Suggests that social interaction alone is not stressful.  A state of arousal induced by exposure to an appetitive stimulus (receptive female) has no effect on the reinstatement of heroin seeking.  Suggests that the neurochemistry of arousal from a social interaction might be the same as a negative stressor but yields distinguishable behavioral outcomes.  An encounter with a non-aggressive opponent does not reinstate CPP.  Suggests that social interaction alone is not stressful.  A state of arousal induced by exposure to an appetitive stimulus (receptive female) has no effect on the reinstatement of heroin seeking.  Suggests that the neurochemistry of arousal from a social interaction might be the same as a negative stressor but yields distinguishable behavioral outcomes. Discussion

 Exposure to social stress produces the same effects as physical stressors on the reinstatement of morphine- induced CPP.  Mechanisms involved are yet to be elucidated.  Further conclusions?  Exposure to social stress produces the same effects as physical stressors on the reinstatement of morphine- induced CPP.  Mechanisms involved are yet to be elucidated.  Further conclusions? Conclusion

THE END Thank you Andrew R. Burke Thank you Andrew R. Burke